Dihydroxyproply-cysteine peptide and agent containing this peptide

ABSTRACT

The invention relates to a S-(2,3-dihydroxypropyl)-cysteine peptide which has two long-chain fatty acids bonded in the form of esters at the dihydroxypropyl group, and which has the following sequence: 
     
       
         
               
               
             
                   
                 (SEQ ID NO: 1) 
               
                   
                 DhcGN NDE SNI SFK EK. 
               
           
              
              
             
          
         
       
     
     The invention relates also to a composition comprising the mentioned peptide.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No. 10/385,098 filed Mar. 10, 2003 entitled “Dihydroxypropyl-Cysteine Peptide and Agent Containing this Peptide” which is a division of U.S. patent application Ser. No. 09/331,087 filed Jun. 16, 1999, now U.S. Pat. No. 6,573,242 B1 granted Jun. 3, 2003, which is a 371 filing of international patent application PCT/EP97/07090 filed Dec. 17, 1997 which claims priority benefits of German Patent Application DE 1 96 52 586.1 filed Dec. 17, 1996, the disclosures of all are hereby incorporated by reference.

The macrophage-stimulating activity of mycoplasma has been known for a relatively long time; see Loewenstein at al. in Cellular Immunology, 77 (1983) 290-297. It has also been assumed and formally proved that lipoproteins from mycoplasma exhibit such an activity; see Herbelin at al. in Infect. Immun., 62 (1994) 4690-4694 and Mühlradt at al. in Biochemistry, 35 (1996) 7781-7786. Lipoproteins from Gram-negative bacteria and analogues of those lipoproteins are likewise immunomodulators and have been described specifically as macrophage activators; see Melchers et al. in J. Exp. Med., 142 (1975) 473-482 and Hoffmann et al. in Immunobiol., 177 (1988) 158-170. Those species of lipoproteins carry an N-terminal S-(2,3-dihydroxy-propyl)-cysteine group (Dhc) having three long-chain fatty acids, of which two are bonded in the form of esters and one is bonded in the form of an amide.

Lipoproteins and synthetic lipopeptide analogues have a half-maximum effective concentration (Max/2) of approximately 10⁻⁷ M; see Melchers at al. in J. Exp. Med., 142 (1975) 473-482 and Hoffmann et at in Biol. Chem. Hoppe Seyler, 370 (1989) 575-582.

Synthetic analogues without the amide fatty acid have a half-maximum effective concentration (Max/2) of approximately 18⁻⁸ M; see Metzger at al. in J. Peptide Scie., 3 (1995) 184-190. Furthermore, in Tetrahedron, 45 (1989) 6331-6360, 6352, Baschang described a taurine-modified lipopeptide (sodium sulphonate: CGP-31 362) which, according to Dong et al. in J. Exp. Med., 177 (1993) 1071-1077, still has macrophage-activating action at from 1 to 10 ng/ml or from 1 to 10×10⁻⁹ M. Finally, in J. Peptide Scie. 3 (1995) 184-190, Metzger et al. describe a Dhc peptide having the amino acid sequence CFE PPP ATT T (SEQ ID NO: 2), two palmitoyl groups being bonded to the 2,3-dihydroxypropyl group. The half-maximum effective concentration (Max/2) of that known peptide is 16 ng/ml or 10×10⁻⁹ M.

There is, however, still a need for effective lipopeptides.

According to the invention there is now proposed a S-(2,3-dihydroxypropyl)-cysteine peptide having two fatty acids, which may be identical or different, bonded to the dihydroxypropyl group in the form of esters, the peptide having the following amino acid sequence (I):

(SEQ ID NO: 1) DhcGN NDE SNI SEK EK (SEQ ID NO: 1) (I) or (SEQ ID NO: 1)         1 Dhcys Gly Asn Asn Asp Glu Ser Asn Ile Ser Phe Lys      13 Glu Lys or an amino acid sequence that is identical to the sequence (I) except that the two N-terminal amino acids in positions 2 and, optionally, 3 are missing and/or one or two C-terminal amino acids have been deleted.

According to the invention, the two fatty acid radicals may have the formula R—CO—, wherein R is a C₇-C₂₅-alkyl, C₇-C₂₅-alkenyl or C₇-C₂₅-alkynyl radical, unsaturated radicals preferably being present in the cis configuration. Examples of C₇-C₂₅-alkyl, -alkenyl and -alkynyl radicals are C₁₆ and C₁₈ radicals.

According to the invention there is also provided a composition comprising a S-(2,3-dihydroxypropyl)-cysteine peptide according to the invention together with a conventional carrier and/or adjuvant. The composition according to the invention can be used for stimulating the synthesis of antibodies, for preventing infections (anti-infective activity), as an immunostimulant against tumours, for activating macrophages, for developing tolerance towards endotoxins or in the case of septic shock, especially in the case of Gram-negative bacteria, or as a vaccine adjuvant (admixture with a vaccine).

According to the invention, S-(2,3-dihydroxypropyl)-cysteine peptides can be prepared in a fully synthetic manner. The person skilled in the art can proceed analogously to the cited prior art. Reference is made also to DE 35 46 150 A1, DE 37 00 173 A1, DE 38 13 821 A1, DE 41 19 856 A1 and DE 43 29 309 A1.

The invention is explained in greater detail below with reference to an Example.

EXAMPLE

The lipopeptide is prepared from Mycoplasma fermentans (for example PG18). The isolation of the lipopeptide from mycoplasma is carried out by the following known separation procedure (Mühlradt et al. in Biochemistry, 35 (1996) 7781-7786).

(i) Delipidation of the mycoplasma using chloroform/methanol.

(ii) Extraction of the delipidated mycoplasma using hot 25 mM octyl glucoside.

(iii) Dialysis of the detergent extract.

(iv) Concentration of the extract by lyophilisation.

(v) Reversed-phase chromatography on a C8 column using a water/2-propanol gradient.

Detection of the biological activity is effected by measuring nitrite and nitrate as the secondary products of nitrogen monoxide, which is liberated on stimulation of interferon-treated murine peritoneal macrophages.

The active ingredient is a S-(2,3-dihydroxypropyl)-cysteine peptide which has two long-chain fatty acids (C16:0 and C18:0/C18:1) bonded in the form of esters at the dihydroxypropyl group, and which has the following sequence:

(SEQ ID NO: 1) Dhc-GNN DES NIS FKE K

The most frequent molecular weight is 2164. In addition, it is possible to find variants which are distinguished by different fatty acids and by a peptide that is shortened by two C-terminal amino acids.

The substance has the property of stimulating macrophages of mice and humans to release cytokines and prostaglandins, with all the consequences of indirect stimulation of T and B lymphocytes; see Mühlradt et al. in Infect. Immun., 59 (1991) 3962-3968 and Feng & Lo in Infect. Immun., 62 (1994) 3916-3921. Its half-maximum effective concentration (Max/2) is 20 pg/ml or 10⁻¹¹ M in the mouse system. That effective concentration is lower by a factor of from 10² to 10³ than the corresponding known concentrations of similar natural or synthetic lipopeptides. 

1. S-(2,3-Dihydroxypropyl)-cysteine peptide having two fluty acids, which may be identical or different, bonded in the form of esters to the dihdroxypropyl group, the peptide having the following ammo acid sequence (I): DheGN NDE SNI SFK EK (SEQ ID NO: 1) or an amino acid sequence that is identical to sequence (I) except that the N-terminal amino acids in positions 2 and, optionally, 3 are missing and/or one or two C-terminal ammo acids have been deleted.
 2. S42,3-Dihydroxypropyl)-cysteine peptide according to claim 1, wherein that the two fatty acid radicals have the formula R—CO—, wherein R is a C₇-C₂₅-alkyl, C₇-C₂₅-alkenyl or C₇-C₂₅-alkynyl group, unsaturated radicals preferably being present in the cis configuration.
 3. Composition comprising a S-(2,3-dihydroxypropyl)-cysteine peptide according to claim 1 together with a conventional carrier and/or adjuvant.
 4. Composition according to claim 3 for stimulating the synthesis of antibodies.
 5. Composition according to claim 3 for preventing infections (anti-infective activity).
 6. Composition according to claim 3 for immunostimulation against tumours.
 7. Composition according to claim 3 for activating macrophages.
 8. Composition according to claim 3 for developing tolerance towards endotoxins (septic shock), especially in the case of Gram-negative bacteria.
 9. Composition according to claim 3 as a vaccine adjuvant (admixture with a vaccine).
 10. Composition according to claim 3 for short-term protection against infections, especially Crowding disease, in animals, especially livestock, working animals or domestic animals. 